Fluorescent protein development 1) We have an ongoing project to develop improved green photoactivatable fluorescent proteins (PAFPs) for Photoactivated Localization Microscopy (PALM). PALM is a super-resolution technique, which accesses information below the diffraction barrier of most optical microscopy techniques by localizing sparse subsets of molecules with high precision using two-dimensional Gaussian fits of their diffraction-limited fluorescence signals (spot size of 500 nm diameter). PAFPs make possible this technique by having molecules that are dark before a pulse of light turns on their fluorescence. This allows densely populated specimens to be imaged one or just a few molecules at a time, necessary for the molecular localization described above. We have also embarked on a project to develop a photoactivatable version of a new fluorescent protein, Clover, which has high brightness and may alleviate some of the brightness problem in our PAGFP variants. 2) We have a study of the little known green-to-red photoconversion which occurs in the commonly used EGFP variant. Numerous reports dating back to the late 1990s have found that different forms of the green fluorescent protein can photoconvert into a red fluorescent species during intense irradiation. Initially it was reported that low oxygen conditions were required, but a similar phenomenon was noted in the presence of oxidizing agents. Our own studies have found that EGFP can undergo a green-to-red photoconversion in aerated samples lacking oxidizing agents, suggesting it is an inherent property of the protein. A survey of FP variants has found one which does not undergo this process. Comparison of the primary structures shows several differences and by performing single point mutagenesis at those positions the green-to-red photoconversion has been recovered. Now we are targeting the amino acid position for mutagenesis in EGFP of interest to both inhibit photoconversion as well as enhance this phenomenon. As second part of this project is to analyze the photobleaching behavior (loss in green fluorescence) in concert with redding effect. The effect of point mutants around the chromophore is being used to map the influence of various amino acids as well as the specific regions of influence.. 3) We collaborate with Joy Zhao and Peter Schuck on their development of new fluorescence ultracentrifugation techniques. As a consequence, we have commenced a project to survey numerous fluorescent proteins to better define their oligomerization characteristics. Our surveys of the literature have suggested these characteristics have not been rigorously determined using the proper ultracentrifugation analysis. Our hypothesis is that aberrant behavior observed when these proteins are tagged to some proteins of interest may be due to oligomerization. Cell biology projects 1) We collaborate with Anamaris Colberg-Poley on super-resolution imaging of human cytomegalovirus infected cells. Our interest is gaining insight into the transfer of pUL37x1 protein from mitochondria associated membranes to the outer mitochondria membrane. Data have been collected on single expressed proteins using uninfected cells expressing PAFP tagged versions of the pUL37x1. Ongoing work is shifting to 2-color imaging to compare the localizations of multiple proteins of interest. This project has been transferred to Ernesto Casillas. 2) With Raul Rojas, a staff scientist in the lab of Lawrence Tabak, we have undertaken a project to image with PALM the localization of Golgi apparatus enzymes. These studies are intended to help in our understanding of where the enzymes are located within the Golgi and what role these locations may play in the enzymatic activity. Specifically, we are interested in whether enzymes involved in early steps in sugar modifications of proteins are located in early compartments of the Golgi and vice versa. We have performed preliminary experiments using a marker consisting of the localization domain of galactosyltransferase and the photoactivatable fluorescent protein, PAmCherry. Raul has just finished developing plasmids containing the chimeras with the full length enzymes. In addition, we are working to determine the centers of mass for the proteins of interest in nococazole induced Golgi fragments to give indications of their relative positions within Golgi mini-stacks. This work is done in collaboration with Maria Ingaramo. Instrumentation and imaging development 1) Two-photon excitation on our PALM system has been upgraded using a microlens array to produce a grid pattern of diffraction limited spots. By rapidly scanning the pattern (100Hz) using a galvanometer, a wider field of view (compared to the temporal focus) can be evenly illuminated. This addition has two advantages. First, it allows the user to change the wavelength of two-photon excitation with little realignment of the laser beam. Additionally, this upgrade allows us to implement a new super-resolution structured illumination microscopy (SIM) technique, multi-focal SIM, developed by the Shroff lab. This instrument represents the next phase in MSIM development by incorporating multi-photon excitation. This allows super-resolution imaging in tissue at depths unattainable by current super-resolution techniques. 2) A total internal reflection fluorescence (TIRF) microscope system has been built by Yan Fu on an Olympus IX70 microscope obtained through property transfer. This instrument is been designed to provide a more homogeneous illumination pattern compared with traditional through objective TIRF by illuminating the sample from all angles possible for a through objective configuration. This is achieved by rapidly scanning the excitation beam in a circular pattern around the periphery of the objective rear aperture using galvanometers. By doing so, any diffraction patterns or other aberrations often found in TIRF imaging are averaged by imaging from various angles. We have developed a method to image at multiple positions with the TIRF excitation zone which effectively allows optical sections at 20-50 nm increment through the 200-300 nm illumination region. 3) We collaborated with Andy York and Hari Shroff on the development of a new image processing technique relying on joint deconvolution of multiple images with various strengths which complement and overcome their various weaknesses. With this method, images with high signal-to-noise but low resolution are deconvolved jointly with images of low signal but high resolution to produce an image with high S/N and high resolution. This approach has two key features which make it of interest to the wider community. First, the same method can be used for multiple imaging modes, such as PALM, Structured Illumination Microscopy (SIM), and gated Stimulated Emission Depletion Microscopy (gSTED). The only difference is the illumination pattern that must be input into the processing code. Second, the method requires no user input other than the illumination pattern and the number of iterations. Thus, it reduces user errors in over or under processing images.